Factor ix polypeptide mutant, its uses and a method for its production

ABSTRACT

Disclosed are a modified FIX (Factor IX) polypeptide comprising a leucine, cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine or tyrosine in position 338; pharmaceutical preparations containing said modified FIX polypeptide; a nucleotide sequence coding for the modified FIX polypeptide; and a method for producing the modified FIX polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a is a continuation of U.S. patent application Ser.No. 16/787,456 filed Feb. 11, 2020, which is a continuation of U.S.patent application Ser. No. 16/707,414 filed Dec. 9, 2019, which is acontinuation of U.S. patent application Ser. No. 16/589,851 filed Oct.1, 2019, which is a continuation of U.S. patent application Ser. No.15/989,665 filed May 25, 2018 now U.S. Pat. No. 10,465,180 issued Nov.5, 2019, which is a continuation of U.S. patent application Ser. No.15/650,070 filed Jul. 14, 2017, now U.S. Pat. No. 9,982,248 issued May29, 2018, which is a continuation of U.S. patent application Ser. No.14/981,981 filed Dec. 29, 2015, now abandoned, which is a continuationof U.S. patent application Ser. No. 13/063,898 filed May 31, 2011, nowU.S. Pat. No. 9,249,405 issued Feb. 2, 2016, which is a 371 nationalstage of International Application No. PCT/EP2009/061935 filed Sep. 15,2009, which claim the benefit of Italian Application Nos. BO2009A000275filed May 6, 2009 and BO2008A000564 filed Sep. 15, 2008. All of theforegoing are incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a modified AX (factor IX) polypeptide,a nucleotide sequence, a vector comprising said nucleotide sequence anda method for producing the modified AX polypeptide.

The present invention also relates to pharmaceutical preparations anduses of modified factor FIX and of the nucleotide sequence.

PRIOR ART

FIX is a vitamin K-dependent glycoprotein belonging to theserine-protease family, and is synthesized in the liver of man and otheranimals, including mammals, playing a fundamental role in both intrinsicand extrinsic pathways of the coagulation cascade. Human FIX circulatesin plasma as a single chain zymogen composed of 415 amino acids. HumanFIX has a molecular weight of 56 kD and a plasma concentration of about5 pg/ml. The zymogen is activated both by activated factor XI (FXIa),and tissue factor complex (TF)—activated factor VII (FVIIa). Thestructural organization of FIX is similar to that of other vitaminK-dependent coagulation proteins such as factor VII (FV1I), factor X(FX) and protein C (PC). The amino-terminal portion of the moleculecomprises the “Gla” domain, a region rich in gamma-carboxy-glutamicresidues whose carboxylation is dependent on the presence of vitamin K.The main physiological function of FIX, once activated, is to convertfactor X (FX) into activated factor X (FXa) in a process that requiresthe presence of a phospholipid surface, calcium ions and a protein withcofactor effect, namely activated factor VIII (FVIIIa). FXa itself isable to convert prothrombin into thrombin which transforms fibrinogeninto soluble fibrin which, on polymerization, forms the clot. The actionof FXa is enhanced by the presence of activated factor V (FVa).

The human FIX gene is located on chromosome X in position Xq27.1 andcontains 8 exons of lengths varying from 25 base pairs (bp) to 2000 bp.Human FIX mRNA is about 3 kb in length and comprises 205 bases whichform the 5′ UTR region, 1386 bases which encode the FIX polypeptide and1392 bases of the 3′ UTR region. This mRNA encodes the synthesis of 461amino acids which form the human FIX precursor. This precursor (SEQ IDNO: 1) comprises the following segments and domains: a hydrophobicsignal peptide (amino acids 1-28), a propeptide (amino acids 29-46), aGla-domain (amino acids 47 to 92), an EGF-like 1 domain (amino acids 93to 129), an EGF-like 2 domain (amino acids 130 to 171), an activationpeptide (amino acids 192 to 226) and a serine-protease domain (aminoacids 227 to 461). The mature form of human FIX (SEQ ID NO: 2) loses thehydrophobic signal peptide and the propeptide. Consequently thecorresponding amino acid positions of the aforementioned domains becomethe following: a Gla-domain (amino acids 1 to 46), an EGF-like 1 domain(amino acids 47 to 83), an EGF-like 2 domain (amino acids 84 to 125), anactivation peptide (amino acids 146 to 180) and a serine-protease domain(amino acids 181 to 415). SEQ ID NO: 1 (from which SEQ ID NO: 2 isderived) corresponds to the sequence on PubMed (“Protein” category)found by entering accession number AAB59620; this amino acid sequencecomprises the signal peptide (46 AA), followed by the amino acidsequence of the mature protein.

A genetic deficiency in FIX can cause a number of coagulation diseases(coagulopathies), for example the haemorrhagic disease known ashaemophilia B in affected males (sex linked genetic disease).Haemophilia B can be classified into three classes, each of which ischaracterized by the presence of different plasma concentrations of FIX.In severe haemophilia B the plasma levels of FIX activity are below 1%of normal; in the moderate form, levels are between 1% and 5%; in themild form, between 5 and 25% of normal levels. There are also healthycarrier individuals who have medium FIX activity levels, between 25% and50% of normal, but many carriers can have levels even exceeding 50%.Patients affected by severe haemophilia B present serious haemorrhagicmanifestations which can be controlled or avoided by administering FIXconcentrates of extractive (from human plasma) or of recombinant origin,currently only available in a single commercial formulation.

Attempts to correct the genetic defect by means of gene therapy have sofar been fruitless because of various problems. These include firstlythose connected to the low efficiency of expression in man of FIX levelsin plasma i.e. around 1%, hence not sufficient to correct the disease;those connected to the immunogenicity of treatment with viral vectors;finally those connected to the side effects of gene therapy itself whichinclude hepatitis, myositis and others.

An increase in plasma FIX to higher than normal levels (normal range ofFIX in plasma being 70-120% i.e. 70-120 U/dl, where a unit is thequantity of FIX contained in 1 millilitre of normal plasma, equal toabout 5 μg) has been associated with an increased risk in humans ofdeveloping thrombotic manifestations in the venous system. Inparticular, for values above 150 U/dl, a 4.8 fold increase in thromboticrisk has been noted (corrected 0. R. 4.8; 95% CI, from 2.3 to 10.1).However, the genetic basis for the increased FIX levels in plasma ofthese individuals has never been identified.

In vitro mutagenesis studies of mutated recombinant FIX expression havedemonstrated the possibility of reproducing the alterations in FIXsynthesis and activity encountered in vivo in patients with haemophiliaB. Vice-versa, by site-specific mutagenesis in certain positions on theFIX molecule, FIX mutants have been produced with “gain-of-function”(increased activity relative to the normal molecule) by altering theirspecificity for physiological substrates and/or modifying their otherfunctions. In WO 99/03496 is disclosed the recombinant FIX arginine 338alanine mutant which resulted in a gain-of-function whose activitylevels are 2-3 folds higher than that found in wild type FIX. Thesegain-of-function mutants (in particular with increased protease activitytowards the physiological substrate, i.e. FX, or with an increasedcapacity for interaction with FV111a, a cofactor of FIXa) have not asyet been found to exist in nature, nor have they been tested in man.More explicitly, there is no evidence of: 1) the existence of a humancarrier of mutated FIX (natural FIX mutant) with gain-of-functioncharacterized by increased functional activity as compared to normal FIX(WT) with any gain-of-function in functional activity; 2) testsconducted in vivo in man with administrations of modified recombinantFIX; 3) tests conducted in vivo in man with administrations of modifiedrecombinant FIX with gain-of-function for the prophylaxis and treatmentof patients affected by haemophilia (genetic or acquired) or othercoagulopathies; 4) tests conducted in vivo in man with administrationsof modified recombinant FIX which show the absence of side effects.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a modified FIXpolypeptide, a nucleotide sequence, a vector comprising said nucleotidesequence, and a method for producing the modified FIX polypeptide.

A further object of the present invention is to provide pharmaceuticalpreparations and uses for modified factor FIX and the nucleotidesequence.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention are provided polypeptides, nucleotidesequences, vectors, a method of production, uses of the polypeptides andnucleotide sequences and the pharmaceutical preparations, according tothat described in the following independent claims and preferably in anyone of the claims that depend directly or indirectly on the independentclaims.

The modified FIX polypeptides herein described show a gain-of-functionof at least 5 folds higher than that of the wild-type FIX molecule. Thisincrease in the activity level is unexpectedly even higher than thatdisclosed for the known recombinant FIX arginine 338 alanine mutant.

The contents of the references (articles, textbooks, GenBank sequencesetc.) cited in the present text are fully included herein fordescriptive completion. In particular, the references (articles,textbooks, GenBank sequences etc.) cited in the present text areincorporated herein for reference. Unless otherwise explicitlyspecified, the following terms have the meanings indicated below.

In the present text the term “percentage identity” and “% identity”between two amino acid (peptide) or nucleic acid (nucleotide) sequencesmeans the percentage of identical amino acid or nucleotide residues incorresponding positions in the two optimally aligned sequences.

To determine the “percentage identity” of the two amino acid or nucleicacid sequences, the sequences are aligned together. To achieve anoptimal match, gaps can be introduced into the sequence (i.e. deletionsor insertions which can also be placed at the sequence ends). Amino acidand nucleotide residues in the corresponding positions are thencompared. When a position in the first sequence is occupied by the sameamino acid or nucleotide residue that occupies the correspondingposition in the second sequence, the molecules are identical in thatposition. The percentage identity between two sequences is a function ofthe number of identical positions divided by the sequences [i.e. %identity=(number of identical positions/total number of positions)×100]

According to an advantageous embodiment, the sequences have the samelength. Advantageously, the compared sequences do not have gaps (orinsertions).

The percentage identity can be obtained by using mathematicalalgorithms. A non-limiting example of an algorithm used for comparingtwo sequences is the Karlin and Altschul algorithm [Proc. Natl. Acad.Sci. USA 87 (1990) 2264-2268] modified by Karlin and Altschul [Proc.Natl. Acad. Sci. USA 90 (1993) 5873-5877]. Said algorithm isincorporated in the BLASTn and BLASTp programmes of Altschul [Altschulet al, J. Mol. Bio. 215 (1990) 403-410].

With the purpose of achieving alignments even in the presence of one ormore gaps (or insertions) methods may be used which assign a relativelyhigh penalty for each gap (or insertion) and a lower penalty for eachadditional amino acid or nucleotide residue in the gap (this additionalamino acid or nucleotide residue is defined as gap extension). Highpenalties will obviously lead to the alignments being optimized with theleast number of gaps.

An example of a programme able to achieve this type of alignment is theBLAST programme as described in Altschul et al., Nucleic Acids Res. 25(1997) 3389-3402. For this purpose the BLASTn and BLASTp programmes canbe used with the default parameters. When using the BLAST programme theBLOSUM62 matrix is typically employed.

An advantageous and non-limiting example of a programme for achieving anoptimal alignment is GCG Wisconsin Bestfit package (University ofWisconsin, USA; Devereux et. al., 1984, Nucleic Acid Research 12:387).The default parameters are again used i.e. for an amino acid sequencethey allow a penalty of −12 for a gap and a penalty of −4 for eachextension.

In the present text the term “percentage homology” and “% homology”between two amino acid or nucleotide sequences means the percentage ofhomologous amino acid or nucleotide residues in corresponding positionsin the two optimally aligned sequences.

The percentage homology between two sequences is determined in asubstantially identical manner to that described above for determiningpercentage identity except for the fact that homologous positions andnot only identical positions are considered in the calculation.

With regard to a nucleotide sequence, two homologous positions presenttwo different nucleotides but which, within their codon, code for thesame amino acid. With regard to an amino acid sequence, two homologouspositions present two homologous amino acids, that is to say amino acidspossessing similar physicochemical properties, for example amino acidsbelonging to the same groups such as: aromatic (Phe, Trp, Tyr), acids(Glu, Asp), polar (Gln, Asn), basic (Lys, Arg, His), aliphatic (Ala,Leu, Ile, Val), with a hydroxyl group (Ser, Thr), with a short sidechain (Gly, Ala, Ser, Thr, Met). It is expected that substitutionsbetween these homologous amino acids would not change the phenotype ofthe proteins (conservative amino acid substitutions). Specific examplesof conservative substitutions are known in this technical field and aredescribed in the various literature (e.g. Bowie et al., Science,247:1306-1310 (1990)).

Further examples of programmes and/or articles relating to thedetermination of alignments and percentage homologies and/or identitiesare indicated in, for example, US2008003202, US2007093443, WO06048777.

In the present text the term “corresponding position” means a positionin a polypeptide or nucleic acid sequence which, following an alignment,corresponds to (or faces), a precise position in a reference sequence.For example, a position corresponding to a precise position on the FIXpolypeptide presenting SEQ ID NO: 2 can be determined by aligning theSEQ ID NO: 2 with a polypeptide of interest; the alignment can becarried out manually or as explained above in relation to percentageidentity determination.

In the present text the term “naked chain” means a polypeptide which hasnot been chemically modified but contains only covalently bound aminoacids.

In the present text the term “promoter” means a DNA portion of a genethat controls (activates) the transcription of a nucleotide sequence towhich it is operatively linked (but not necessarily flanking it). Thepromoter includes one or more DNA sequences, which are recognized by RNApolymerase and bind RNA polymerase so that RNA polymerase itselfinitiates transcription.

In the present text the term “treat” or “treatment” of a pathology meansthe prophylaxis and/or therapy and/or cure of this pathology. The termprophylaxis means advantageously to at least partially arrest thedevelopment of a potential disease and/or to prevent the worsening ofsymptoms or progression of a disease. Advantageously, the term therapymeans a partial or total alleviation of the disease symptoms.

In the present text the term “vector” means an element used to introducea nucleic acid into a cell for the expression or replication of saidnucleic acid. An example of vectors are episomes, which are capable ofextra-chromosomal replication. The vectors can also be integrated intohost chromosomes. Vectors are often in the form of plasmids, generallycircular double-helical DNA.

In the present text “vehicle presenting a nucleic acid” means: a vectorwhich includes nucleic acid; a cell which includes nucleic acid; or apharmaceutically acceptable excipient combined with the nucleic acid bymixing. Advantageously the vehicle is chosen from a vector or a cell.

The invention will now be described with reference to the accompanyingdrawing which illustrates a non-limiting example of its implementation,in which:

FIG. 1 illustrates an SDS-PAGE and immunoblot of a normal FIXpolypeptide (2), a modified FIX polypeptide according to the presentinvention (3), a recombinant modified FIX polypeptide according to thepresent invention (4).

According to a first aspect of the present invention, a modified FIXpolypeptide is provided comprising an amino acid chosen from the groupconsisting of: leucine, cysteine, aspartic acid. glutamic acid.histidine, lysine, asparagine, glutamine, tyrosine in a positioncorresponding to position 338.

According to other embodiments, the amino acid is chosen from the groupconsisting of: leucine, aspartic acid, glutamine.

According to other embodiments, the amino acid is chosen from the groupconsisting of: aspartic acid, glutamine.

According to other embodiments, the amino acid is aspartic acid.

According to other embodiments, the amino acid is glutamine.

According to other embodiments, the amino acid is chosen from the groupconsisting of: aspartic acid, leucine.

According to other embodiments, the amino acid is chosen from the groupconsisting of: leucine, glutamine.

According to other embodiments, the amino acid is leucine.

The modified FIX polypeptide must be able to carry out its functionwithin the coagulation cascade and can be of synthetic or naturalorigin, for example human or animal origin.

Examples of FIX polypeptides include (but are not limited to) unmodifiedwild-type FIX (such as the polypeptide of SEQ ID NO: 2), precursors ofsaid wild-type FIX (such as the polypeptide of SEQ ID NO: 1), naturalpolymorphic variants (such as: a polypeptide presenting an alanine in aposition corresponding to position T148 or to a precursor polypeptidethereof).

In the present text the loci (positions) of the modified or unmodifiedamino acid sequences are identified by reference to the amino acidnumbering in the corresponding positions of an unmodified mature FIXpolypeptide, as identified by SEQ ID NO: 2. Corresponding positions canbe determined by alignment of unmodified residues (see above). By way ofexample we report hereinafter the sequences and relative numberings ofthe mature FIX polypeptide (SEQ ID NO: 2) and of the FIX polypeptideprecursor (SEQ ID NO:1).

SEQ ID NO: 1 MQRVNMIMAE SPGLITICLL GYLLSAECTV FLDHENANKILNRPKRYNSG KLEEFVQGNL ERECMEEKCS FEEAREVFENTERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCPFGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEGYRLAENQKSC EPAVPFPCGR VSVSQTSKLT RAEAVFPDVDYVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPWQVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAGEHNIEETEHT EQKRNVIRII PHHNYNAAIN KYNHDIALLELDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF HKGRSALVLQ YLRVPLVDRA TCL RSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE GTSFLTGIIS WGEECAMKGKYGIYTKVSRY VNWIKEKTKL Tin bold underlined Arg 384 corresponding to Arg 338 in SEQ ID NO: 2

SEQ ID NO: 2 YNSGKLEEFV QGNLERECME EKCSFEEARE VFENTERTTEFWKQYVDGDQ CESNPCLNGG SCKDDINSYE CWCPFGFEGKNCELDVTCNI KNGRCEQFCK NSADNKVVCS CTEGYRLAENQKSCEPAVPF PCGRVSVSQT SKLTRAETVF PDVDYVNSTEAETILDNITQ STQSFNDFTR VVGGEDAKPG QFPWQVVLNGKVDAFCGGSI VNEKWIVTAA HCVETGVKIT VVAGEHNIEETEHTEQKRNV IRIIPHHNYN AAINKYNHDI ALLELDEPLVLNSYVTPICI ADKEYTNIFL KFGSGYVSGW GRVFHKGRSA LVLQYLRVPL VDRATCL RST KFTIYNNMFC AGFHEGGRDS CQGDSGGPHV TEVEGTSFLT GIISWGEECA MKGKYGIYTKVSRYVNWIKE KTKLTin bold underlined Arg 338.

Likewise, the positions of the modified or unmodified nucleotidesequences are identified, unless otherwise indicated, by reference tothe nucleotide numbering in the corresponding positions of thenucleotide sequence identified by accession number K02402 (GenBank). Thenucleotide sequence K02402 codes for the AX polypeptide precursor (SEQID NO: 1) and includes some intron regions (in this regard see Anson DS, Choo K H, Rees D J, Giannelli F, Gould K, Huddleston J A, Brownlee GG. The gene structure of human anti-haemophilic factor IX. The EMBOJournal 1984; 3:1053-1060).

Included within the definition of a modified FIX polypeptide arechimeric variants which can be produced by replacing amino acids orentire domains of the FIX with amino acids or sequences of other factorsbelonging to the coagulation factor family (for example factor VII orfactor X). According to other embodiments, the modified FIX polypeptidepresented herein is either a naked chain or exhibitspost-transcriptional modifications. Examples of modifications includeone or more chemical modifications, which comprise (but are not limitedto): glycosylation, acylation, methylation, phosphorylation, sulphation,carboxylation, salification, vitamin C-dependent modifications such ashydrolysis of proline, aspartic acid, lysine, or carboxy-terminalamidation; vitamin K-dependent modifications such as carboxylation ofglutamic acid residues; incorporation of selenium to form one or moreselenocysteine(s); incorporation of a PEG moiety (polyethylene glycol).

In addition to the possible modifications disclosed herein, the modifiedFIX polypeptide can contain one or more variants known in the state ofthe art such as hyperglycosylation, deimmunization and others (see forexample: U.S. Pat. Nos. 6,277,618, 6,315,995, 6,531,298, US2004/0102388,US2004/0110675, US2004/0254106, 052005/0100982, US2006/0040856).Non-limiting examples of modified FIX polypeptide variants can bededuced from one or more of the following references: US2006/040856,Friedler et al (2000) J. Biol Chem. 275:23783-23789, US2004/102388,WO2006/018201, Lim et al. (1990) J. Biol Chem. 265(1):144-150, Cheung etal. (1992) J. Biol. Chem. 267(29): 20529-20531, Gui et al. (2002) Blood100(1)1 53-158, Schuettrumpf et al. (2005) Blood 105(6): 2316-2323,US2004/110675, U.S. Pat. No. 6,315,995.

According to some alternative embodiments, the modified FIX polypeptidehas at least 50%, 60%, 70%, 80%, 85%, 90%, 94%, 97%, 99%, 100% homology(or, advantageously, identity) with a peptide sequence chosen from thegroup consisting of: SEQ ID NO: 1 and SEQ ID NO: 2.

Advantageously, the modified FIX polypeptide has at least 60% homology(or, advantageously, identity) with a peptide sequence chosen from thegroup consisting of: SEQ ID NO: 1 and SEQ ID NO: 2.

Advantageously, the modified FIX polypeptide has at least 80% homology(or, advantageously, identity) with a peptide sequence chosen from thegroup consisting of: SEQ ID NO: 1 and SEQ ID NO: 2.

Advantageously, the modified FIX polypeptide has at least 90% homology(or, advantageously, identity) with a peptide sequence chosen from thegroup consisting of: SEQ ID NO: 1 and SEQ ID NO: 2.

Advantageously, the peptide sequence is SEQ ID NO: 2.

According to a second aspect of the present invention, a nucleotidesequence is provided which codes for the FIX polypeptide of the firstaspect of the present invention.

According to some alternative embodiments, the nucleotide sequence hasat least 50%, 60%, 70%, 80%, 85%, 90%, 94%, 97%, 99%, 100% homology (or,advantageously, identity) with the sequence having accession numberK02402 (GenBank).

Advantageously, the nucleotide sequence has at least 70% homology (or,advantageously, identity) with the sequence having accession numberK02402 (GenBank).

Advantageously, the nucleotide sequence has at least 90% homology (or,identity) with the sequence having accession number K02402 (GenBank).

Advantageously, the nucleotide sequence has at least 100% homology (or,advantageously, identity) with the sequence having accession numberK02402 (GenBank).

According to some alternative embodiments, the nucleotide sequence is aRNA sequence and has at least 50%, 60%, 70%, 80%, 85%, 90%, 94%, 97%,99%, 100% homology (or, advantageously, identity) with the sequence fromposition 31 to position 1411 (SEQ ID NO: 3) (advantageously fromposition 169 to position 1411-SEQ ID NO: 4) of the polynucleotide ofFIG. 2 in the article by Anson D S, Choo K H, Rees D J, Giannelli F,Gould K, Huddleston J A and Brownlee G G. The gene structure of humananti-hemophilic factor IX. The EMBO Journal 1984; 3: 1053-1060. In thiscase (that is, with reference to SEQ ID NO: 3 and SEQ ID NO: 4), theposition numbers refer to the numbering reported in the aforementionedFIG. 2. Advantageously, the RNA sequence has at least 80% homology (or,advantageously, identity) with the sequence SEQ ID NO: 3(advantageously, SEQ ID NO: 4). Advantageously, the RNA sequence has atleast 90% homology (or, advantageously, identity) with the sequence SEQID NO: 3 (advantageously, SEQ ID NO: 4). Advantageously, the RNAsequence has at least 95% homology (or, advantageously, identity) withthe sequence SEQ ID NO: 3 (advantageously, SEQ ID NO: 4).

The RNA sequence can be linked, at the head and/or tail, to additionalnucleotide chains that are either not translated or translatedseparately.

According to some alternative embodiments, the nucleotide sequence is aDNA sequence and comprises (in particular, consists of) intron and exonportions, which present an overall sequence (that is to say exonportions without gaps and linked together in order) having at least 50%,60%, 70%, 80%, 85%, 90%, 94%, 97%, 99%, 100% homology (or,advantageously, identity) with the overall sequence of exon regions inthe sequence (SEQ ID NO: 5) of FIG. 4 in the article by Anson D S, ChooK H, Rees D J, Giannelli F, Gould K, Huddleston J A and Brownlee G G.The gene structure of human anti-hemophilic factor IX. The EMBO Journal1984; 3:1053-1060.

Advantageously, the exon portions are separate from each other andplaced in order (arranged relative to each other) as are the respectiveexon regions in the sequence SEQ ID NO: 5. Advantageously, the overallsequence of the exon portions has at least 80% homology (or,advantageously, identity) with the overall sequence of the exon regions.Advantageously, the overall sequence of the exon portions has at least90% homology (or, advantageously, identity) with the overall sequence ofthe exon regions. Advantageously, the overall sequence of the exonportions has at least 95% homology (or, advantageously, identity) withthe overall sequence of the exon regions.

According to some embodiments, the nucleotide sequence comprises athymine in a position corresponding to position 34099 (or in thecorresponding position 32318 according to the numbering given in SEQ IDNO: 5; or in the corresponding position 31134 according to the numberinggiven in the Database of mutations of Hemophilia B (Giannelli et al.,Hemophilia B: Database of point mutations and short additions anddeletions. Nucleic Acids Research 1990; 18:4053-9); or a uracil in thecorresponding position 11180 of SEQ ID NO: 3 or SEQ ID NO: 4).

In other words, the aforementioned nucleotide sequence differs from thesequence having accession number K02402 (GenBank) by at least the factof bearing a mutation from guanine to thymine in position 34099(G34099T) or in a corresponding position (for example position 32318according to the numbering of SEQ ID NO: 5; or from guanine to uracil inthe corresponding position 11180 of SEQ ID NO: 3 or SEQ ID NO: 4).

In this case the nucleotide sequence codes for a leucine in a positioncorresponding to position 338.

According to some embodiments, the nucleotide sequence in the positionscorresponding to 34098, 34099 and 34100, presents a triplet chosen fromthe group consisting of: TTA, UUA, TTG, UUG, CTT, CUU, CTC, CUC, CTA,CUA, CTG, CUG, GAT, GAU, GAC, CAA, CAG. In particular, when thenucleotide sequence is a DNA sequence, the triplet is chosen from thegroup consisting of TTA, TTG, CTT, CTC, CTA, CTG, GAT, GAC, CAA, CAG.

According to some embodiments, the nucleotide sequence, in the positionscorresponding to 34098, 34099 and 34100, presents a triplet chosen fromthe group consisting of: TTA, UUA, TTG, UUG, CTT, CUU, CTC, CUC, CTA,CUA, CTG, CUG, CAA, CAG. In particular, when the nucleotide sequence isa DNA sequence, the triplet is chosen from the group consisting of TTA,TTG, CTT, CTC, CTA, CTG, CAA, CAG. In these cases, the sequence codesfor a leucine or a glutamine in a position corresponding to position338.

According to some embodiments, the nucleotide sequence, in the positionscorresponding to 34098, 34099 and 34100, presents a triplet chosen fromthe group consisting of: TTA, UUA, TTG, UUG, CTT, CUU, CTC, CUC, CTA,CUA, CTG, CUG. In particular, when the nucleotide sequence is a DNAsequence, the triplet is chosen from the group consisting of TTA, TTG,CTT, CTC, CTA, CTG. Advantageously, the triplet is CTA. In these cases,the sequence codes for a leucine in a position corresponding to position338.

According to some embodiments, the nucleotide sequence, in the positionscorresponding to 34098, 34099 and 34100, presents a triplet chosen fromthe group consisting of: CAA, CAG. In these cases, the sequence codesfor a glutamine in a position corresponding to position 338.Advantageously, the triplet is CAA. To obtain the CAA triplet, anadenine is inserted in place of the guanine in position 34099.

According to some embodiments, the nucleotide sequence, in the positionscorresponding to 34098, 34099 and 34100, presents a triplet chosen fromthe group consisting of: GAT, GAU, GAC, CAA, CAG. In particular, whenthe nucleotide sequence is a DNA sequence, the triplet is chosen fromthe group consisting of GAT, GAC, CAA, CAG. In these cases, the sequencecodes for an aspartic acid or a glutamine in a position corresponding toposition 338. According to some embodiments, the nucleotide sequence, inthe positions corresponding to 34098, 34099 and 34100, presents atriplet chosen from the group consisting of: GAT, GAU, GAC. Inparticular, when the nucleotide sequence is a DNA sequence, the tripletis chosen from the group consisting of GAT, GAC. In these cases, thesequence codes for an aspartic acid in a position corresponding toposition 338. Advantageously, the triplet is GAT. To obtain the GATtriplet, a guanine is inserted in place of the adenine in position34098, an adenine in place of the guanine in position 34099 and athymine in place of the adenine in position 34100.

The aforesaid homology (or identity) percentages are calculated withoutconsidering the specific mutated positions indicated. In other words,for example, the sequence SEQ ID NO: 2 modified with a leucine inposition 338 is considered as having 100% homology (and identity) withthe unmodified sequence SEQ ID NO: 2.

According to a third aspect of the present invention, a nucleic acid isprovided which comprises a nucleotide sequence according to the secondaspect of the present invention.

According to some embodiments, the nucleic acid comprises a promoter inoperational linkage with the nucleotide sequence.

According to a fourth aspect of the present invention, a vector isprovided comprising a nucleic acid as aforedefined in relation to thethird aspect of the present invention. In particular, the vectorcomprises a nucleotide sequence according to the second aspect of thepresent invention.

According to some embodiments, the vector is chosen from: a prokaryotevector, a eukaryote vector or a viral vector.

Advantageously, the vector is a viral vector. In particular, the vectoris chosen from: an adenovirus, a retrovirus, a herpesvirus, alentivirus, a poxvirus, a cytomegalovirus.

According to a fifth aspect of the present invention, a method for theproduction of a modified FIX polypeptide is provided, whereby themodified FIX polypeptide is expressed by means of a nucleic acidaccording to the third aspect of the present invention.

According to some embodiments the method comprises the steps of:introducing a vector of the fourth aspect of the present invention intoa cell; and culturing the cell such that the FIX polypeptide isexpressed.

Alternatively, the modified FIX polypeptide can be produced by a hostanimal or in vitro from the aforementioned nucleotide sequence.

According to a particular aspect of the present invention, the methodcomprises the steps of: introducing the nucleotide sequence of thesecond aspect of the present invention into a cell-free system;expressing the modified polypeptide in the cell-free system.

According to a further particular aspect of the present invention, themethod allows the modified FIX polypeptide to be expressed in atransgenic animal comprising a nucleic acid in accordance with the thirdaspect of the present invention (in particular, the nucleotide sequenceof the second aspect of the present invention). Useful hosts forexpression of the modified FIX polypeptide include: E. coli, yeasts,plants, insect cells, mammalian cells (Pham et al. (2003) Biotechnol.Bioeng. 84:332-42; Bon et al. (1998) Semin Hematol. 35 (2 Suppl 2):11-17; Wahij et al., J. Biol. Chem. 280 (36) 31603-31607) and transgenicanimals.

The hosts can vary as to their levels of protein production and also thetypes of modifications induced in the modified FIX polypeptidesubsequent to transcription. Eukaryote hosts can include yeasts such asSaccharomyces cerevisiae and Pichia pastoris (Skoko et al. (2003)Biotechnol. Appl. Biochem. 38 (Pt 3): 257-65), insect cells (Muneta etal. (2003) J. Vet. Med. Sci. 65(2): 219-23), plants and cells fromplants such as tobacco, rice, algae (Mayfield et al. (2003) PNAS100:438-442) etc. The plants are typically modified by direct transferof DNA and agrobacterium-mediated transformations. Advantageously usablevectors comprise promoter sequences and transcription termination andcontrol elements.

Yeasts are usually modified by replicating episomal vectors or by astable chromosomal integration by homologous recombination.Advantageously, promoters are used to regulate gene expression. Examplesof promoters include GAL1, GALT, GALS, CUP1. Proteins produced by yeastsare usually soluble; alternatively, proteins expressed in yeasts can besecreted.

Expression in eukaryotic hosts also includes production in animals, forexample in serum, milk and eggs. Transgenic animals for the productionof FIX polypeptides are known (for example US2002/0166130 andUS2004/0133930) and can be adapted for producing the modified FIXpolypeptide as aforedefined.

Prokaryote cells in particular E. coli can be advantageously utilized toproduce large quantities of modified FIX polypeptide as aforedefined(Platis et al. (2003) Protein Exp. Purif. 31(2):222-30; Khalizzadeh etal. (2004) J. Ind. Microbial. Biotechnol. 31(2): 63-69).

The vectors used with E. coli advantageously contain promoters able toinduce high levels of protein expression and to express proteins thatshow some toxicity towards the host cells. Examples of promoters are T7and SP6 RNA.

Reducing agents such as β-mercaptoethanol can be utilized to solubilisepolypeptides which may precipitate in the cytoplasmic environment of E.coli.

According to a sixth aspect of the present invention, a modified FIXpolypeptide is also provided in accordance with the first aspect of thepresent invention, for use as a medicament.

The modified FIX polypeptide can be used for disease treatments eitheralone or in combination with other active compounds.

The modified FIX polypeptide is useful for treating coagulopathies(congenital or acquired), haematological diseases (congenital oracquired), haemorrhagic disorders (such as haemorrhagic gastritis and/oruterine bleeding), other cardiovascular diseases.

According to some embodiments, the modified FIX polypeptide is providedfor the treatment of at least one coagulopathy.

According to some embodiments, the modified FIX polypeptide is providedfor the treatment of haematological diseases.

According to some embodiments, the modified FIX polypeptide is providedfor the treatment of haemorrhagic disorders.

According to some embodiments, the modified FIX polypeptide isadministered to patients periodically for relatively long time periodsor before, during and/or after surgical procedures to reduce and/orprevent haemorrhages.

The use of modified FIX polypeptide for the treatment of coagulopathiesis particularly effective. Advantageously, modified FIX polypeptide isused for the treatment of haemophilia, and in particular haemophilia Aand haemophilia B.

According to advantageous embodiments, the modified FIX polypeptide isprovided for treating haemophilia B, and advantageously severe and/ormoderate haemophilia B.

Advantageously, modified FIX polypeptide is used for the treatment ofmammals, in particular human patients.

According to a seventh and an eighth aspect of the present invention,the following are provided: use of the modified FIX polypeptide inaccordance with the first aspect of the present invention for preparinga drug (pharmaceutical preparation) advantageously for treating acoagulopathy; and a pharmaceutical preparation comprising the modifiedFIX polypeptide and, advantageously, at least one pharmaceuticallyacceptable excipient.

According to some embodiments, the pharmaceutical preparation is for thetreatment of a pathology chosen from the group consisting of:coagulopathies (congenital or acquired), haematological diseases(congenital or acquired), haemorrhagic disorders (such as haemorrhagicgastritis and/or uterine bleeding), haemophilia (haemophilia A orhaemophilia B). According to specific embodiments, the pharmaceuticalpreparation is for treating a coagulopathy. According to specificembodiments, the pharmaceutical preparation is for treating haemophilia.

According to a further aspect of the present invention, a method isprovided for treating at least one coagulopathy, this method allowingthe administration of an effective quantity of a modified FIXpolypeptide as aforedefined.

The modified FIX polypeptide can be administered as a pure compound, butis advantageously presented in the form of a pharmaceutical preparation.Non-limiting examples of pharmaceutical preparations if needed for thispurpose are explained below.

The modified FIX polypeptide can be formulated for oral, parenteral orrectal administration, or in forms suited to administrations byinhalation or insufflation (either via the mouth or nose). Formulationsfor oral or parenteral administration are advantageous.

For oral administrations, the pharmaceutical preparations are in theform of, for example, tablets or capsules prepared by known methods withpharmaceutically acceptable excipients such as binders (for examplepregelatinized maize starch, polyvinylpyrrolidone, or methyl cellulose);fillers (for example lactose, microcrystalline cellulose or calciumhydrogen phosphate); additives (for example magnesium stearate, talc,silica); disintegrants (for example potato starch); and/or lubricants(for example sodium lauryl sulphate). The tablets can be coated usingknown methods. Liquid preparations for oral administration have theform, for example, of solutions, syrups or suspensions, or can be in theform of a dry product that can be dissolved in water or another liquidprior to use. Said preparations are prepared by known methods withpharmaceutically acceptable additives such as suspending agents (forexample sorbitol, cellulose derivatives, edible hydrogenated fats);emulsifying agents (for example lecithin or acacia); non-aqueous liquids(for example almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and/or preservatives (for example methyl orpropyl-hydroxybenzoates, sorbic acid or ascorbic acid). The preparationscan also contain, in appropriate cases, buffering salts, colouringagents, flavouring agents and/or sweeteners.

Preparations for oral administration are formulated in a known manner,in order to provide a controlled release of the active compound.

The modified FIX polypeptide is formulated, in a known manner, forparenteral administration, by injection or continuous administration.Formulations for injection are, advantageously, in the form of dosageunits, for example in ampoules or multi-dose containers containingpreservatives. The composition can be in the form of a suspension, inaqueous or oily liquids, and can contain elements of the formulation asdispersing and stabilizing agents. Alternatively, the active compoundcan be in powder form to be dissolved just before use in a liquid asneeded. such as sterile water.

The modified FIX polypeptide can be formulated for rectal administrationas suppositories or enemas, for example, containing suppositoryexcipients of known type such as cocoa butter or other glycerides.

The modified FIX polypeptide is also formulated, in a known manner, inextended release compositions. These extended release compositions are,for example, administered by means of an implant (for examplesubcutaneous or intramuscular) or an intramuscular injection. Therefore,for example, the modified FIX polypeptide is formulated with suitablepolymer or hydrophobic materials (such as an emulsion or an oil) or ionexchange resins, or relatively poorly soluble derivatives, such asrelatively poorly soluble salts.

For intranasal administration, the modified FIX polypeptide isformulated by administrations via a (known) device, such as in a powderwith a suitable vehicle. The dosages of the modified FIX polypeptidewill depend on the patient age and condition, and so the precise dosagewill have to be decided each time by the physician. The dosage will alsodepend on the mode of administration and the particular compoundselected. Usable doses can be for example comprised between 0.1 mg/kgand 400 mg/kg body weight per day.

According to a further aspect of the present invention, the nucleotidesequence is provided in accordance with the second aspect of the presentinvention for use as a medicament (advantageously for treating acoagulopathy).

The nucleotide sequence can be used for treating a pathology eitheralone or in combination with other active compounds.

The nucleotide sequence is useful for treating the pathologies of thesixth aspect of the present invention.

According to particular aspects of the present invention, the followingare provided: the use of the aforementioned nucleotide sequence forpreparing a drug advantageously for treating a coagulopathy; and apharmaceutical preparation containing the nucleotide sequence.

Instead of administering the modified FIX polypeptide it is possible toadminister the nucleotide sequence which encodes it.

The nucleotide sequence can be inserted into cells or tissues by meansof any known method. The nucleotide sequence can be incorporated into avector for subsequent manipulations.

For example, certain cells could be engineered so as to express themodified FIX polypeptide, by integrating the aforementioned nucleotidesequence into a genomic location operatively linked with the promotersequences. Said cells can be administered to a patient locally orsystemically. Usable viral vectors include poxvirus, herpesvirus,retrovirus, adenovirus, adeno-associated virus and other virusessuitable for gene therapy.

The vectors can remain as episomal or can be integrated into thechromosomes of the treated individual. Adenovirus serotypes arecommercially available from the American Type Culture Collection (ATCC,Rockville).

The viral vectors, in particular adenovirus, are used ex vivo; forexample, cells are isolated from a patient and transduced with anadenovirus expressing the modified FIX polypeptide. After a suitableperiod of culturing, the transduced cells are administered to thepatient locally or systemically.

Alternatively, the viruses, in particular adenoviruses, which expressthe modified FIX polypeptide are isolated and formulated with apharmaceutically acceptable excipient and administered to the patient.Typically, the adenoviruses are administered at doses of 1 to 1014particles per kilogram of patient weight, generally from 106 to 1012particles per kilogram of patient weight.

Additional examples of cell types for the expression and release of themodified FIX polypeptide are fibroblasts and endothelial cells (Palmeret al. (1989) Blood 73:483-445; Yao et al (1991) PNAS 88:8101-8105).

A vehicle which presents the aforementioned nucleotide sequence can beformulated in a similar manner to that described above for the modifiedFIX polypeptide.

The nucleotide sequence and/or drugs and/or vehicles presenting saidnucleotide sequence can be used for treating the pathologies referred toabove in relation to the modified FIX peptide. Advantageously, theaforementioned nucleotide sequence is used for treating mammals, inparticular human patients.

According to a further aspect of the present invention, a method isprovided for detecting the protein of the first aspect of the presentinvention and/or the nucleotide sequence of the second aspect of thepresent invention.

Usable methods are those known in the state of the art, and can beadapted to those polymorphism under study to include for exampleimmunoenzymatic assays, coagulation protein activity tests (includingFIX activity), coagulometric and chromogenic tests.

According to some embodiments, the method comprises a step of amplifyingby PCR part of a nucleic acid molecule (in which it is required toverify the presence of the nucleotide sequence of the second aspect ofthe present invention).

Advantageously, the amplification step is preceded by a step ofpurifying, in particular isolating, the nucleic acid molecule.

Advantageously the amplification step is followed by a sequencing step.

By way of example, the methods of examples 2 and 3 below can be followedto detect the aforesaid nucleotide sequence.

The method for detecting the protein and/or nucleotide sequence can beused to assist in the identification of those individuals who display ahigh tendency to develop blood diseases such as thrombosis.

Further characteristics of the present invention will ensue from thefollowing description of some examples which are merely illustrative andnon-limiting.

Example 1—Routine Laboratory Tests Carried Out on the Proband

Routine laboratory coagulation tests were carried out with regard tothrombophilia screening on an individual (defined as the Proband)exhibiting episodes of deep vein thrombosis but no other healthproblems.

In particular, the following were carried out: prothrombin time, partialthromboplastin time, factor IX levels, factor VIII and X1 levels,antithrombin levels (activity and antigen), protein C levels(coagulometric and chromogenic activity, antigen), protein S levels(total antigen, free antigen and activity), activated protein Cresistance, DNA analysis for factor V Leiden, DNA analysis for theprothrombin variant G20210A, antiphospholipid antibodies, plasminogen,fibrinolysis tests. The coagulation tests carried out on the Probandwere all found to be within normal limits except for FIX activity (seeexample 4 below).

Example 2—Isolation of Mutant FIX from Plasma and from the Cell CultureMedium

Isolation of FIX from plasma or from culture medium was achieved bymeans of the immunoaffinity column technique, using a resin (sepharose4B) to which the anti-FIX monoclonal antibody AHIX-5041 [HaematologicTechnologies, Inc. (Essex Junction, Vt., USA)] was covalently bound (3.5mg of monoclonal antibody per 3 ml of sepharose resin). Briefly, thecolumn was equilibrated with buffer containing 20 mM Tris, 150 mM NaCl,1 mM benzamidine (mM=millimolar). Starting from the plasma, vitaminK-dependent factors were precipitated by adding barium chloride. Aftercentrifugation, the sediment was resuspended in a solution containing0.2 M EDTA. The preparation thus obtained was extensively dialyzed (2times, for at least 2 hours) in a solution containing 20 mM Tris, 150 mMNaCl. After dialysis, the preparation was permitted to pass through thecolumn at a rate of 0.5 ml/min. After extensive column washing (10column volumes) with Tris/NaCl buffer, elution was carried out using asolution of acidic glycine (pH 2.45). The eluate pH was immediatelyneutralized by adding 2 M Tris at pH 7.5. The eluate fractionscontaining protein (tested by the Bradford protein assay) were pooledand dialyzed against a Tris-NaCl solution, the FIX was then concentratedthrough a 2000 microcolumn of fast-flow sepharose Q (ion exchange). Thepurity of the preparation was evaluated by applying the silver stainingtechnique on the SDS-PAGE gel.

Example 3—Genetic Study of FIX

PCR amplification and direct sequencing of the exons and splice sites ofthe Proband FIX gene were carried out using standardized techniques andprimers as reported in the literature (From: Methods in MolecularMedicine, Vol 31: Hemostasis and Thrombosis Protocols. Edited by D. J.Perry and K. J. Pasi. Humana Press Inc. Totowa, N.J. Chapter 16:Hemophilia B mutational analysis. By Peter Green). Briefly,amplification was carried out by using intron primer pairs flanking eachof the eight exons of the FIX gene. The sequencing was undertaken withan ABI PRISM 310 sequencer (Perkin Elmer, Foster City, Calif.) using theABI PRISM BigDye Terminator kit for cycle sequencing reactions. Thesequence data were analyzed using the Sequencing Analysis 3.0 programme(Perkin Elmer, CA). The sequence obtained was compared with the FIXsequence reported on the GenBank database (accession number: K02402).

Analysis of the nucleotide sequence of the Proband FIX gene hasdocumented a single mutation in exon VIII of the FIX gene compared tothe normal sequence. The patient was found to be a carrier for amutation from G to T at position 34099 of the FIX gene (normal sequenceof the FIX gene, Gene bank accession number: K02402) (or in thecorresponding position 31134 according to the numbering given in theDatabase of mutations of Hemophilia B (Giannelli et al., Hemophilia B:Database of point mutations and short additions and deletions. NucleicAcids Research 1990; 18:4053-9) able to change codon 338 from Arginineto Leucine. Therefore the FIX molecule present in the Proband's plasma(mutated FIX) differs from the normal FIX molecule only by the presenceof the amino acid substitution in position 338 where there is a Leucineinstead of Arginine.

Example 4—In Vitro Mutagenesis, Expression and Purification ofRecombinant FIX Containing the Leu 338 Mutation

Site-specific mutagenesis was carried out according to standardtechniques described by Kunkel (Kunkel TA. Rapid and efficientsite-specific mutagenesis without phenotypic selection; Proc Nati AcadSci, USA 1985, 82:488-492). Sequencing of the cDNA was carried out forassurance that the mutation was correct and that any new mutations hadnot been introduced. Expression of the recombinant FIX was obtainedusing “human embryonic kidney cell line 293” and the methods alreadyreported in the literature (Chang J L, Jin J P, Lollar P, et al.Changing residue 338 in human factor IX from arginine to alanine causesan increase in catalytic activity. J. Biol. Chem. 1998;273:12089-12094). The recombinant FIX was isolated from the supernatant(culture medium) by means of an immunoaffinity column, asaforedescribed. Briefly, the supernatant of the cell culture wascollected every 24 hours for 10 days and conserved at −20²C. For thepurification the supernatant was thawed out and benzamidine and EDTAwere added to a final concentration of 5 milliMoles and 4 milliMolesrespectively. After filtration through a Millipore filter, thesupernatant was incubated with fast-flow Sepharose Q resin for 12 hoursat 4° C. The resin was then re-equilibrated in Tris, NaCl andbenzamidine buffer and loaded onto the column. Elution was undertakenwith a 0-60 nM calcium gradient. The eluate was then dialyzed in aTris-NaCl buffer. The preparation was then applied to the immunoaffinitycolumn following the method described in example 2 (in the “in vitro”expression of the recombinant protein). Starting from the culturemedium, the procedure was the same as for the plasma, except for theprecipitation procedure using BaCI. The culture medium was centrifugedat 4000 g for 20 minutes then subjected to dialysis in Tris-NaCl andloaded onto the immunoaffinity column at a rate of 0.5 ml/min.

The remaining steps were the same as those taken for the plasma.

The FIX with the G34099T gene mutation resulting in the 338Leu aminoacid substitution, was obtained by in vitro mutagenesis and expressiontechniques. The level of expression in cell culture was found to besimilar to that obtainable with non-mutated recombinant FIX (normalmolecule). Specifically, the expression level of the non-mutatedrecombinant FIX was between 750 and 880 ng/ml while for the recombinantfactor IX with the gene mutation G340991 resulting in the 338Leu aminoacid substitution, the level was between 590 and 629 ng/ml.

Example 5—Functional Assay of FIX

The functional assay of FIX was carried out on the Proband's plasma witha coagulometric test using Actin (Dade Behring, Marburg, Germany) andFIX deficient plasma (Dade Behring, Marburg, Germany). Briefly for thecoagulometric test a variant of the partial thromboplastin time (PTT)was used in a system containing FIX deficient plasma. After adding thecalcium chloride the clotting time was measured in seconds. Thisclotting time was compared to those of a calibration curve obtained byserial dilutions of a pool of normal plasma as reference containing FIXat a quantity of 5 μg/ml (i.e. 100%), and the FIX percentage present inthe sample being calculated on 100% of the normal plasma pool (accordingto common standardized methods).

The normal range for the test had been previously obtained by analyzing,using the same method, 100 healthy individuals of both sexes, agedbetween 20 and 70 years.

The activity levels of FIX in the Proband were found to be equal to 776%(normal range in 100 healthy individuals, 80-120%).

Example 6—Assay of FIX Antigen

The FIX antigen was determined with the ELISA test using a firstanti-FIX monoclonal antibody (Affinity Biologicals, Ontario, Canada)coated (bound) onto the plate for the capture and a second monoclonalantibody labelled with Horseradish peroxidase (HRP) (AffinityBiologicals, Canada) for the detection of FIX. The reference curve wasconstructed by diluting a pool of normal plasma from 1:100 to 1:3200 ina buffer for the samples, according to standardized procedures. Briefly,the first antibody was bound to the plate after dilution in sodiumbicarbonate buffer at basic pH (pH=9.0) at a final concentration of 4μg/ml. After extensive washing of the plate with Tris-NaCl-Tween20buffer, the samples, diluted 1:100 and 1:200 in the same buffer, wereloaded into the wells and incubated at ambient temperature for 2 hours.After removal of the samples from the wells and extensive washing withthe buffer, 100 μl of a solution containing the second antibodyconjugated with HRP were added to each of the wells and incubated atambient temperature for two hours. After further washes, 100 μl of asolution containing tetramethylbenzidine (TMB) were added and thedeveloped color was measured by spectrophotometer with a 450 nanometerfilter. The level of FIX antigen was calculated using the referencecurve and expressed as a percentage of the pool of normal plasma. Thenormal test range was previously obtained using the same method byanalyzing 100 healthy individuals of both sexes, aged between 20 and 70years.

FIX antigen levels were found to be equal to 92% (normal range 80-120%).This result (combined with that obtained in example 5) was compatiblewith the presence of normal quantities of a synthesized circulating FIX,but with its procoagulant function being around 8-9 times greater thanthe normal FIX molecule.

Example 7—Activity and Antigen Levels of FIX after Reconstitution of aFIX Deficient Plasma with an FIX Extracted from the Proband's Plasma andwith Recombinant FIX

After isolating FIX from the Proband's plasma, this FIX was used forreconstituting a FIX deficient plasma (Dade-Behring, Milan, Italy) witha final FIX concentration of 5 μg/ml (equal to 100% of normal). Themeasurements of FIX activity and antigen in the thus reconstitutedplasma were 740% and 95% respectively, these being hence comparable withthose of the Proband's plasma. For assaying the activity of therecombinant FIX obtained in accordance with example 4, the same systemwas used after recomposition of a FIX deficient plasma with a quantityof mutated recombinant FIX (rFIX 338Leu) such as to restore the normalFIX concentration in normal human plasma, i.e. 5 μg/ml (corresponding to100% of normal) (μg=micrograms). The measurements of recombinant factorIX activity and antigens were 780% and 90% respectively, these beinghence comparable with those of the Proband's plasma. This indicates thatthe recombinant protein thus obtained, containing the amino acidsubstitution also present in factor IX of the Proband, has a biologicalactivity at least 8-9 times greater than normal factor IX.

Example 8—SDS-PAGE and Immunoblotting of FIX

The SDS-PAGE and immunoblotting (Western blot) of the FIX was carriedout on a 5-15% linear gradient gel according to standard procedures.Briefly, the samples containing normal FIX or recombinant FIX wereloaded into the polyacrylamide gel wells and subjected toelectrophoresis.

The FIX was then subjected to transblotting on a polyvinylidene fluoride(PVDF) membrane using a semidry apparatus (Novablot, GE-Healthcare,Milan, Italy).

The FIX was detected on the PVDF membrane after transblotting using ananti-FIX monoclonal antibody conjugated to HRP (Affinity Biologicals,Ontario, Canada).

FIG. 1 shows that the FIX isolated from the Proband, the 338Leurecombinant FIX and the normal FIX exhibit the same electrophoreticmobility and the same immunoblot pattern (in FIG. 1, 1 indicatesmolecular weight markers, 2 indicates normal FIX, 3 indicates naturalmodified FIX, 4 indicates the modified recombinant FIX).

Therefore no significant differences (neither quantitative norqualitative) between normal human FIX, 338 Leu natural mutant human FIXand 338Leu recombinant FIX were found using this technique.

From the aforedescribed, it is clear that the presence of a leucine in aposition corresponding to position 338 surprisingly increases theactivity of FIX polypeptide by almost eight times.

The present invention proves to be a particular improvement on the stateof the art as it provides a modified FIX polypeptide which in vivo inman does not cause any side effects other than an increased coagulationactivity.

Example 9—In Vitro Mutagenesis, Expression and Purification of theRecombinant FIX Containing the 338 Asp Mutation (338 Aspartic Acid,338D)

The site-specific mutagenesis was carried out according to standardtechniques described by Kunkel (Kunkel TA. Rapid and efficientsite-specific mutagenesis without phenotypic selection; Proc Nati AcadSci USA 1985, 82: 488-492) by inserting a guanine in place of cytosinein position 34098, and an alanine in place of guanine in position 34099and a thymine in place of alanine in position 34100 (the mutagenesis wasalso repeated by inserting a guanine in place of cytosine in position34098, an adenine in place of guanine in position 34099 and a guanine inplace of adenine in position 34100).

Sequencing of the cDNA was carried out for assurance that the mutationwas correct and that any new mutations had not been introduced.Expression of the recombinant FIX was obtained using “human embryonickidney cell line 293” and the methods already reported in the literature(Chang J L, Jin J P, Lollar P, et al. Changing residue 338 in humanfactor IX from arginine to alanine causes an increase in catalyticactivity. J. Biol. Chem. 1998; 273:12089-12094). The recombinant FIX wasisolated from the supernatant (culture medium) by means of animmunoaffinity column, as aforedescribed. Briefly, the supernatant ofthe cell culture was collected every 24 hours for 10 days and conservedat −20° C. For the purification the supernatant was thawed out andbenzamidine and EDTA were added to a final concentration of 5 milliMolesand 4 milliMoles respectively. After filtration through a Milliporefilter, the supernatant was incubated with fast-flow Sepharose Q resinfor 12 hours at 4° C. The resin was then re-equilibrated in Tris, NaCland benzamidine buffer and loaded onto the column. Elution wasundertaken with a 0-60 nM calcium gradient. The eluate was then dialyzedin a Tris-NaCl buffer. The preparation was applied to the immunoaffinitycolumn following the method described in example 2 (in the “in vitro”expression of the recombinant protein). Starting from the culturemedium, the procedure was the same as for the plasma, except for theprecipitation procedure using BaCI. The culture medium was centrifugedat 4000 g for 20 minutes then subjected to dialysis in Tris-NaCl andloaded onto the immunoaffinity column at a rate of 0.5 ml/min. Theremaining steps were the same as those taken for the plasma.

The FIX with the amino acid substitution 338Asp was obtained by in vitromutagenesis and expression techniques. The level of expression in cellculture was found to be similar to that obtainable with non-mutatedrecombinant FIX (normal molecule). Specifically, the expression level ofthe non-mutated recombinant FIX was between 750 and 880 ng/ml while forthe recombinant factor IX with the 338Asp amino acid substitution, thelevel was between 650 and 740 ng/ml.

Example 10—Activity and Antigen Levels of FIX after Reconstitution of aFIX Deficient Plasma with Recombinant FIX with 338Asp Mutation

For the assay of the activity of recombinant FIX obtained in accordancewith example 9, the same system was used after recomposition of a FIXdeficient plasma with a quantity of mutated recombinant FIX (rFIX338Asp) such as to restore the normal concentration of FIX in normalhuman plasma, i.e. 5 μg/ml (corresponding to 100% of normal)(μg=micrograms). Measurements of recombinant factor IX activity andantigens were 460% and 98% respectively. This indicates that therecombinant protein thus obtained (FIX 338 Asp), has a biologicalactivity at least 5 times greater than normal factor IX.

Example 11—SDS-PAGE and Immunoblotting of FIX

The SDS-PAGE and immunoblotting (Western blot) of the FIX was carriedout on a 5-15% linear gradient gel according to standard procedures.Briefly, the samples containing normal FIX or recombinant FIX wereloaded into the polyacrylamide gel wells and subjected toelectrophoresis.

The FIX was then subjected to transblotting on a polyvinylidene fluoride(PVDF) membrane using a semidry apparatus (Novablot, GE-Healthcare.Milan, Italy).

The FIX was detected on the PVDF membrane after transblotting using ananti-FIX monoclonal antibody conjugated to HRP (Affinity Biologicals,Ontario, Canada).

The 338Asp recombinant FIX and the normal FIX exhibit the sameelectrophoretic mobility and the same immunoblot pattern. Therefore nosignificant differences (neither quantitative nor qualitative) betweennormal human FIX and 338Asp recombinant FIX were found using thistechnique.

From the aforedescribed, it is clear that the presence of an Asparticacid in a position corresponding to position 338 surprisingly increasesthe activity of FIX polypeptide by almost eight times.

The present invention proves to be a particular improvement on the stateof the art as it provides a modified FIX polypeptide which in vivo inman does not cause any side effects other than an increased coagulationactivity.

Example 12—In Vitro Mutagenesis, Expression and Purification ofRecombinant FIX Containing the 338GIn Mutation (338 Glutamine, 3380)

Site-specific mutagenesis was carried out according to standardtechniques described by Kunkel (Kunkel TA. Rapid and efficientsite-specific mutagenesis without phenotypic selection; Proc Nati AcadSci USA 1985, 82: 488-492) by inserting an adenine in place of guaninein position 34099 (the mutagenesis was also repeated by inserting anadenine in place of guanine in position 34099, and a guanine in place ofadenine in position 34100). Sequencing of the cDNA was carried out forassurance that the mutation was correct and that any new mutations hadnot been introduced. Expression of the recombinant FIX was obtainedusing “human embryonic kidney cell line 293” and the methods alreadyreported in the literature (Chang J L, Jin J P, Lollar P, et al.Changing residue 338 in human factor IX from arginine to alanine causesan increase in catalytic activity. J. Biol. Chem. 1998;273:12089-12094). The recombinant FIX was isolated from the supernatant(culture medium) by means of an immunoaffinity column, asaforedescribed. Briefly, the supernatant of the cell culture wascollected every 24 hours for 10 days and conserved at −20° C. For thepurification the supernatant was thawed out and benzamidine and EDTAwere added to a final concentration of 5 milliMoles and 4 milliMolesrespectively. After filtration through a Millipore filter, thesupernatant was incubated with fast-flow Sepharose 0 resin for 12 hoursat 4° C. The resin was then re-equilibrated in Tris, NaCl andbenzamidine buffer and loaded onto the column. Elution was undertakenwith a 0-60 nM calcium gradient. The eluate was then dialyzed in aTris-NaCl buffer. The preparation was then applied to the immunoaffinitycolumn following the method described in example 2 (in the “in vitro”expression of the recombinant protein). Starting from the culturemedium, the procedure was the same as for the plasma, except for theprecipitation procedure using BaCI. The culture medium was centrifugedat 4000 g for 20 minutes then subjected to dialysis in Tris-NaCl andloaded onto the immunoaffinity column at a rate of 0.5 ml/min. Theremaining steps were the same as those taken for the plasma.

The FIX with the amino acid substitution 338Gin was obtained by in vitromutagenesis and expression techniques. The level of expression in cellculture was found to be similar to that obtainable with non-mutatedrecombinant FIX (normal molecule). Specifically, the expression level ofthe non-mutated recombinant FIX was between 750 and 880 ng/ml while forthe recombinant factor IX with the 338GIn amino acid substitution, thelevel was between 600 and 720 ng/ml.

Example 13—Levels of Activity and Antigen of the FIX afterReconstitution of a FIX Deficient Plasma with Recombinant FIX with338GIn Mutation

For the assay of the activity of recombinant FIX obtained in accordancewith example 12, the same system was used after recomposition of a FIXdeficient plasma with a quantity of mutated recombinant FIX (rFIX338GIn) such as to restore the normal concentration of FIX in normalhuman plasma, i.e. 5 μg/ml (corresponding to 100% of normal)(μg=micrograms). Measurements of recombinant factor IX activity andantigens were 1360% and 99% respectively. This indicates that therecombinant protein thus obtained (FIX 338 Gin) has a biologicalactivity at least 13 times greater than normal factor IX.

Example 14—SDS-PAGE and Immunoblotting of FIX

The SDS-PAGE and immunoblotting (Western blot) of the FIX was carriedout on a 5-15% linear gradient gel according to standard procedures.Briefly, the samples containing normal FIX or recombinant FIX wereloaded into polyacrylamide gel wells and subjected to electrophoresis.

The FIX was then subjected to transblotting on a polyvinylidene fluoride(PVDF) membrane using a semidry apparatus (Novablot, GE-Healthcare,Milan, Italy).

The FIX was detected on the PVDF membrane after transblotting using ananti-FIX monoclonal antibody conjugated to HRP (Affinity Biologicals,Ontario, Canada).

The 338GIn recombinant FIX and the normal FIX exhibited the sameelectrophoretic mobility and the same immunoblot pattern. Therefore nosignificant differences (neither quantitative nor qualitative) betweennormal human FIX and 338GIn recombinant FIX were found using thistechnique.

From the aforedescribed, it is clear that the presence of a glutamine ina position corresponding to position 338 surprisingly increases theactivity of FIX polypeptide by almost thirteen times.

The present invention proves to be a particular improvement on the stateof the art as it provides a modified FIX polypeptide which in vivo inhumans does not cause any side effects other than an increasedcoagulation activity.

Therefore evidence is provided that:

1) it has been discovered a naturally occurring FIX mutant (arginine 338leucine) with a 8-9 fold increased functional activity as compared toFIX wild-type;2) recombinant modified FIX polypeptides (not known before) with 5 folds(FIX arginine 338 aspartic acid), 8 to 9 folds (FIX arginine 338leucine), 13 folds (FIX arginine 338 glutamine) increased functional(procoagulant) activity, respectively, as compared to FIX wild-type canbe generated.

The use of the mutants of the invention, which show such a specificfunctional activity of 5 folds or above, and in particular 8 to 9 foldsas compared to FIX wild type, for medical use and in particular for theprophylaxis and treatment of Hemophilia B patients; said use of themutants of the invention has never been considered before and is part ofthe present invention.

The use of the mutants of the invention, which show such a specificfunctional activity of 5 folds or above, and in particular 8 to 9 foldsas compared to FIX wild type, for gene therapy of Hemophilia B patientshas never been considered before and is part of the present invention.The use of the mutants of the invention, which show a specificfunctional activity of 5 folds or above, and in particular 8 to 9 foldsas compared to FIX wild type, for the prophylaxis and treatment ofhemorrhagic coagulopathies other than Hemophilia B or for gene therapyof such diseases has never been considered before and is part of thepresent invention.

It has to be noted that the use of the mutants of the invention, whichshow a specific functional activity of 5 folds or above, and inparticular of FIX arginine 338 leucine which shows 8 to 9 foldsincreased functional activity as compared to FIX wild type, isconsidered optimal for the treatment of patients with hemophilia Bbecause of the presence of an identical naturally occurring mutant inhumans (never described before, and is part of the present invention)which does not generate neutralizing antibodies. In addition, the FIXfunctional activity levels express by FIX arginine 338 leucine, ispossibly the best option being higher than that of FIX arginine 338alanine (previously known and described in WO 99/03496, with a modestincrease in activity of 2 to 3 folds that of FIX wild-type) and not toohigh to cause thrombotic complications in hemophilia B patients orpatients with other hemorrhagic coagulopathies.

The invention of FIX arginine 338 leucine, is also the best choice forthe use of FIX mutants in gene therapy by using viral vectors, given theactual efficiency and yield of the method for the treatment (partialcorrection) of Hemophilia B.

According to certain aspects of the present invention there are providedpolypeptides, nucleotide sequences, nucleic acids, vectors, methods anduses in accordance with the following points.

1. A modified FIX (factor IX) polypeptide comprising:an amino acid chosen from the group consisting of: leucine, cysteine,aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine,tyrosine in a position corresponding to position 338.2. A polypeptide according to claim 1 wherein the amino acid is chosenfrom the group consisting of: leucine, aspartic acid. glutamine.3. A polypeptide according to claim 1 wherein the amino acid is chosenfrom the group consisting of: aspartic acid, glutamine.4. A polypeptide according to claim 1 wherein the amino acid is asparticacid.5. A polypeptide according to claim 1 wherein the amino acid isglutamine.6. A polypeptide according to claim 1 wherein the amino acid is leucine.7. A polypeptide according to one of the previous points, and having ahomology of at least 70% with a peptide sequence selected from the groupconsisting of: SEQ ID NO: 1 and SEQ ID NO: 2.8. A polypeptide according to one of the previous points, and having ahomology of at least 90% with a peptide sequence selected from the groupconsisting of: SEQ ID NO: 1 and SEQ ID NO: 2.9. A polypeptide according to one of the previous points, and having apercentage identity of at least 70% with a peptide sequence selectedfrom the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2.10. A polypeptide according to one of the previous points, and having apercentage identity of at least 90% with a peptide sequence selectedfrom the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2.11. A polypeptide according to one of the previous points wherein thepeptide sequence is SEQ ID NO: 2.12. A nucleotide sequence encoding a FIX polypeptide according to one ofthe previous points.13. A nucleotide sequence according to point 12 wherein the nucleotidesequence is a DNA sequence and consists of intron portions and exonportions, the exon portions having an overall sequence with at least 70%homology relative to an overall sequence of exon regions of a SEQ ID NO:5 sequence.14. A nucleotide sequence according to point 13 wherein the overallsequence of the exon portions has at least 90% homology with the overallsequence of the exon regions of the SEQ ID NO: 5 sequence.15. A nucleotide sequence according to one of points 12 to 14 whereinthe nucleotide sequence has at least 50% homology with the sequencehaving the accession number K02402 (Gen Bank).16. A nucleotide sequence according to one of points 12 to 15,comprising in positions corresponding to 34098, 34099 and 34100 atriplet chosen from the group consisting of: TTA, UUA, TTG, UUG, CTT,CUU, CTC, CUC, CTA, CUA, CTG, CUG, GAT, GAU, GAC, CAA, CAG.17. A nucleotide sequence according to point 16, comprising in positionscorresponding to 34098, 34099 and 34100 a triplet chosen from the groupconsisting of: TTA, UUA, TTG, UUG, CTT, CUU, CTC, CUC, CIA, CUA, CTG,CUG, CAA, CAG.18. A nucleotide sequence according to point 16, comprising in positionscorresponding to 34098, 34099 and 34100 a triplet chosen from the groupconsisting of: TTA, UUA, TTG, UUG, CTT, CUU, CTC, CUC, CTA, CUA, CTG,CUG.19. A nucleotide sequence according to point 16, comprising in positionscorresponding to 34098, 34099 and 34100 a triplet chosen from the groupconsisting of: CAA, CAG.20. A nucleotide sequence according to point 16, comprising in positionscorresponding to 34098, 34099 and 34100 a triplet chosen from the groupconsisting of: GAT, GAU, GAC, CAA, CAG.21. A nucleotide sequence according to point 16, comprising in positionscorresponding to 34098, 34099 and 34100 a triplet chosen from the groupconsisting of: GAT, GAU, GAC.22. A nucleotide sequence according to one of points 12 to 18,comprising a thymine in a position corresponding to position 34099.23. A nucleic acid comprising a nucleotide sequence according to one ofpoints 12 to 22.24. A nucleic acid according to point 23, and comprising a promoter inoperational linkage with said nucleotide sequence.25. A vector comprising a nucleic acid according to point 23 or 24.26. A method for producing a modified FIX polypeptide, whereby themodified FIX polypeptide is expressed by means of a nucleic acidaccording to point 23 or 24.27. A method according to point 26, comprising the steps of:introducing a vector of point 25 into a cell; and culturing the cellsuch that the FIX polypeptide is expressed.28. A modified FIX polypeptide according to one of points 1 to 11 foruse as a medicament.29. A modified FIX polypeptide according to one of points 1 to 11 forthe treatment of at least one coagulopathy.30. Use of a modified FIX polypeptide according to one of points 1 to 11for preparing a drug for the treatment of at least one coagulopathy in amammal.31. A nucleotide sequence according to one of points 12 to 22 for use asa medicament.32. A method for detecting the nucleotide sequence of one of points 12to 22.33. A method for detecting the modified FIX polypeptide according to oneof points 1 to 11.34. A method according to point 32 comprising an step of amplificationby PCR.

BIBLIOGRAPHY

-   Ameri A, Kurachi S, Sueishi K, Kuwahara M, Kurachi K. Myocardial    fibrosis in mice with overexpression of human blood coagulation    factor IX. Blood. 2003 Mar. 1; 101 (5):1871-3. Epub 2002 Oct. 24.-   Chang J L, Jin J P, Lollar P, et al. Changing residue 338 in human    factor IX from arginine to alanine causes an increase in catalytic    activity. J Biol Chem 1998; 273:12089-12094.-   Lowe G D O. Factor IX and thrombosis. British Journal of    Haematology, 2001, 115, 507-513.-   Kunkel T A. Rapid and efficient site-specific mutagenesis without    phenotypic selection. Proc Nati Acad Sci USA 1985, 82:488-492.-   Kurachi K, Davie E W. Isolation and characterization of a cDNA    coding for human factor IX. Proc Natl Acad Sci USA 1982;    79:6461-6464.-   Murphy S L, High K A. Gene therapy for haemophilia. Br J Haematol.    2008 March; 140(5):479-87.-   Yoshitake S, Schach B G, Foster D C, et al. Nucleotide Sequence of    thr Gene for Human Factor IX (Antihemophilic Factor B). Biochemistry    1985; 24:3736-3750.-   Toomey J R, Valocik R E, Koster P F, Gabriel M A, McVey M, Hart T K,    Ohlstein E H, Parsons A A, Barone F C. Inhibition of factor IX(a) is    protective in a rat model of thromboembolic stroke. Stroke. 2002    February; 33(2):578-85.

What is claimed is:
 1. An adeno-associated virus (AAV) vectorcomprising: a nucleic acid encoding a modified FIX polypeptide, themodified FIX polypeptide comprising SEQ ID NO:2 with the exception of aleucine at position 338 of SEQ ID NO:2 and an alanine at position 148 ofSEQ ID NO:2.